1. Field of the Invention
This invention is generally in the field of veterinary vaccines, vaccine compositions, and methods of producing the same. Particularly, provided herein are the vaccines for immunizing animal against progressive atrophic rhinitis (PAR), which comprise a combination of at least two fragments of recombinant subunit Pasteurella multocida toxins (rsPMT) each having an amino acid sequence that substantially corresponds to the 2-486, 486-986 or 986-1281 amino acid residues of Pasteurella multocida toxin (PMT).
2. Description of Related Art
Progressive atrophic rhinitis (PAR) is an important upper respiratory disease in swine. The characteristic lesions include turbinate bone hypoplasia, facial distortion and nasal hemorrhage as a result of frequent sneezing. Moreover, PAR causes significant global economic loss in swine production due to growth retardation. Several studies have demonstrated that Pasteurella multocida toxin (PMT) is the major virulence factor responsible for the turbinate atrophy seen in PAR (see, for example, Ackermann M R et al. 1996; Am J Vet Res 57(6):848-852; and Lax A J & Chanter N. 1990; J Gen Microbiol 136:81-87). Inoculation of PMT alone could reproduce all major symptoms of PAR in experimentally challenged pigs. Either aerosolized or injected into swine, PMT causes severe turbinate atrophy and reduces weight gain (Kamp E M & Kimman T G 1988; Am. J. Vet. Res. 49:1844-1849). The mechanisms by which PMT reduces weight gain and conchal bone atrophy have been widely studied. Results of several studies indicate that PMT could increase bone resorption and reduce bone formation by altering the functions of osteoblasts and osteoclasts.
The molecular basis for the virulence of PMT remains unclear, but may be associated with the activation of osteoclasts or inactivation of osteoblasts. It has been demonstrated that PMT is a potent mitogen for several types of cells such as Swiss 3T3 fibroblasts. PMT was able to induce half-maximal stimulation of DNA synthesis and cell proliferation at doses as low as 1 pM. The effect of PMT on porcine osteoclasts and osteoblasts has been investigated using an in vitro cell culture system. Exposure of bone marrow cells to Vitamin D3 and PMT during growth led to an increase in cell numbers and earlier appearance of osteoclasts compared to controls. Low concentrations of PMT resulted in growth retardation and decreased nodule formation in osteoblasts, while high concentrations of PMT increased cell death and inhibited nodule formation (Gwaltney S M et al. 1997; Vet Pathol 34(5):421-430). PMT also stimulates cell proliferation, but impairs cell maturation and cell function in primary cultures of rat osteoblasts. These findings suggest that PMT may increase bone resorption and decrease bone apposition, eventually leading to progressive osteolysis and continuous bone atrophy.
Many potential bacterial pathogens can colonize the nasal cavity or tonsils of swine and P. multocida is one of the primary opportunistic pathogens able to cause porcine respiratory disease complex (PRDC). In fact, PAR is considered a contagious respiratory disease with high prevalence throughout the areas of the world where modern pig husbandry is practiced. Antibiotics, vaccination and good management can reduce the severity and frequency of PAR. However, overuse of antibiotics is a source of public health concern and vaccination has emerged as the most attractive approach in controlling PAR (Foged N T et al. Vet Rec 1989; 125(1):7-11; Kobisch M, Pennings A, Vet Rec 1989; 124(3):57-61; and Sakano T et al. J Vet Med Sci 1997; 59(1):55-57).
The entire PMT gene (toxA) encoding a protein of 1285 amino acids has been cloned and expressed in E. coli (Petersen S K & Foged N T 1989; Infect Immun 57(12):3907-3913). A recombinant PMT derivative lacking N-terminal amino acid residues 27-147 was shown to induce a protective response against challenge with a lethal dose of PMT in mice (Petersen S K et al. 1991; Infect Immun 59(4):1387-1393), and to reduce colonization by toxigenic P. multocida in the nares and tonsils of swine (Nielsen J P et al. 1991; Can J Vet Res 55(2):128-138). Thus, recombinant PMT derivatives may serve as ideal immunogens to elicit a good protective response without cytotoxicity in animals.
Formalin is the most common reagent used to inactivate PMT, but it may induce chemical alterations that can reduce the immunogenicity or efficacy of vaccines (Nielsen J P et al. 1991; as described). Therefore, a non-toxic but immunogenic PMT derivative could be advantageous to the development of effective vaccines against PAR. Most of the PAR vaccines tested to date consist of inactivated cultures of P. multocida or PMT toxoids. The toxoids are prepared by treatment of PMT with formaldehyde, which eliminates toxicity while maintaining antigenicity. These PAR vaccines are effective when tested on farms. However, PMT constitutes less than 0.6% of the total cellular proteins of P. multocida making it necessary to culture a large quantity of bacteria in order to obtain sufficient antigen for commercial scale use. Traditional toxoid vaccines require large scaled culture of a toxigenic strain of P. multocida and a tedious, expensive procedure for preparation of the PMT toxoid. In addition to being both time-consuming and expensive, the need to use inactivating reagents such as formaldehyde may induce uncontrollable chemical alterations in the immunogenicity of proteins that can reduce or eliminate the efficacy of such vaccines.
The N-terminal portion of PMT (residues 1 to 506 a.a.) has been considered to contain the putative cell binding domain and translocation domain. Immunization of the N-terminal rsPMT Tox1 (residues 1 to 487 a.a.) could elicit neutralizing antibodies that could prevent PMT from binding to the target cells and subsequently translocating across the cell membrane. Consequently, the PMT activity was blocked. In addition, the C-terminal portion of PMT was suggested as the catalytic domain and antibodies raised against C-terminal fragments (residues 681-1285 and 849-1285) were capable of inhibiting the mitogenic effect of PMT. The residues 1165 (cysteine), 1205 (histidine) and 1223 (histidine) were showed to be essential for the intracellular activity of PMT (Baldwin M R et al. 2004; 54(1):239-250; and Pullinger G D et al. 2001; Infect. Immun. 69: 7839-7850). The rsPMT derivatives are easy to produce and not cytotoxic so that no extra chemical inactivation is required before use. U.S. Pat. No. 6,110,470 disclosed polypeptide derivatives of P. multocida toxin comprising amino acid sequences identical to PMT but lacking amino acids 1043-1130 or lacking amino acids 1130-1285.
Therefore, in this invention, three recombinant subunit PMT (rsPMT) derivatives, representing the N-terminal (aa. 2-486), middle (aa. 486-986), and C-terminal (aa. 986-1281) portions of PMT, are produced and their immunogenicities are characterized by assessing the level of PMT-specific antibody secreting cells, the serum neutralizing antibody titers and the degree of lymphocyte proliferation in immunized mice and swine. The efficacy of these recombinant subunits as a vaccine was also evaluated in pregnant sows and their offspring by analysis of neutralizing antibody titers in colostrum and serum, and by monitoring the survival rate and the mean weight gain in piglets after PMT challenge.